Alzheimer's disease (AD) is an incurable neurodegenerative disease affecting more than 5 million Americans. Pathologically, extracellular plaques made of the protein amyloid-beta (A) and intracellular tangles made of the protein tau characterize AD. The cause of AD is unknown. However, early changes in patients show that glucose metabolism is altered in affected areas. Specifically, glucose is metabolized preferentially through glycolysis to lactate or through the pentose phosphate pathway rather than continuing on to oxidative phosphorylation, a process termed aerobic glycolysis when oxygen is present. Prior work in cell lines and primary culture further indicates that A leads to increased aerobic glycolysis. Physiologically, neurons have low glycolytic rates and an increase causes apoptosis. This suggests that increased aerobic glycolysis in AD may contribute to neuronal dysfunction and toxicity seen in the disease. We hypothesize that A causes increased aerobic glycolysis, and that this contributes to neuronal dysfunction and the progression of AD. Because many previous studies did not differentiate between neuronal and glial contributions, nor did they examine the effect of metabolic changes at the synapse, we are using tools that allow precise localized measurements. In Aim 1, we will develop an assay to measure aerobic glycolysis with subcellular resolution using a FRET-based glucose sensor, and validated against well-characterized metabolic tracking using mass spectrometry. Our preliminary data demonstrate that we can monitor glucose levels in individual synapses, neuronal cell bodies and glia. Once we have optimized our assay, we will measure A's effect on glucose metabolism in neurons at the cell body and synapse. By controlling glucose uptake and consumption pharmacologically, we will quantify the flux of glucose through aerobic glycolysis and other metabolic pathways to determine how A changes metabolism. In Aim 2, we will delve into the bioenergetic effect of altered aerobic glycolysis to determine how metabolic changes functionally impact neurons. Our lab has developed an assay to measure ATP levels at the synapse. The assay differentiates between mitochondrial and glycolytic- derived ATP using a FRET-based ATP sensor. We will test the effect of A on ATP levels and, functionally, its effect on synaptic transmission with a vGlut1-pHluorin construct. In Aim 3, we will work in vivo to establish whether aerobic glycolysis changes in an AD mouse model and how varying cellular metabolism modifies neuronal function in AD. We will test whether an AD mouse model shows increased aerobic glycolysis using nuclear magnetic resonance spectroscopy. We will then increase aerobic glycolysis in an AD mouse model by knocking out PKM1 in the hippocampus. We will test if increased aerobic glycolysis is detrimental, through behavioral and pathological evaluation. Upon completion of this project, we will have determined whether A increases aerobic glycolysis and how this contributes to toxicity in models of AD, testing a potential therapeutic target. We will have also developed a method to measure glucose metabolism on a single cell level.